Low density lipoproteins (LDL) transport the major portion of plasma cholesterol in humans, and their levels in circulation are directly correlated with the risk for the development of atherosclerotic diseases. Apolipoprotein (apo) B100 is essentially the only apolipoprotein component of LDL and in human liver is an obligatory structural component for the assembly and secretion of triacyglycerol (TAG)-rich very low density lipoproteins (VLDL), the precursors of plasma LDL. ApoB100 also serves as a major ligand for the receptor- mediated uptake of plasma LDL by a variety of cells. Thus, apoB plays a fundamental role in the transport and metabolism of plasma cholesterol and TAG. ApoB is present as a single molecule per particle and therefore, its concentration in the plasma approximates the number of potential atherogenic lipoprotein particles. Because of its pivotal role in the metabolism of atherogenic lipoproteins and as a strong marker for the risk of coronary artery disease (CAD), recently, apoB has been a key therapeutic target for the prevention of cardiovascular diseases. However, current knowledge with respect to the structural elements within apoB polypeptide which are required for the secretion of apoB100, domains that specify recruitment of different lipids, and key motif(s) that mediate TAG addition into the core of the particle is still incomplete. The focus of this application is to delineate th above structure-function relationship of human apoB100 at both the in vitro and in vivo levels. Proposed studies are driven by the following hypotheses: 1) Residues 667-746 in apoB, which are not homologous to any other sequence in lipovitellin or any other sequence in the Genbank database, play an important role in the assembly, structural integrity, and secretion of apoB100-containing lipoproteins in the liver; 2) The intracellular transition of nascent apoB-containing lipoprotein from a PL-rich to a TAG-rich particle requires translation of amino acids beyond residue 1700; 3) The highly conserved 14-residue 1-helix motif in human apoB (residues 124-137) may serve as a key element in the MTP-mediated addition of TAG into the interior of apoB-containing lipoproteins resulting in particle core expansion. These hypotheses will be tested by expression of wild-type and mutant full-length human apoB100 and a carefully selected library of truncated forms of apoB by both in vitro and in vivo approaches, using rat hepatoma McA-RH cells/primary hepatocytes and transgenic mouse models, respectively, by the following 3 Specific Aims. Specific Aim 1. To investigate the roles of the unique domain spanning residues 667-746 in apoB, specifically the helix-loop-helix motif (residues 700-744), and residues 997-1000 at the C-terminus of 211 superdomain as key structural elements in the assembly and secretion of hepatic apoB100-containing particles at both in vitro and in vivo levels. Specific Aim 2. To identify the domains in apoB that recruit various lipids, specifically where the particle transitions from a PL-rich to a TAG-rich particle, and establish the relative roles of PLTP and MTP in this process, using wild-type, MTP- and PLTP-deficient McA-RH cells, and cultured primary hepatocytes. Specific Aim 3. To assess the mechanism of TAG addition to nascent apoB-containing particle by testing the role of the conserved 14-residue 1-helix, residues 124-137 in human apoB, in this MTP- mediated process. This will be achieved by point mutations of key residues in this motif and assessing the subsequent effects on the secretion and composition of the apoB-containing particles both in vitro and in vivo. We will utilize a combination of structural, molecular, cellular, and in vivo approaches to achieve our goals. Proposed studies are aimed at gaining a more in-depth understanding of the structure-function relationship of apoB100. Our overall goal is to exploit these findings toward the development of strategies to attenuate the overproduction of hepatic apoB100-containing lipoproteins, a major cause of hyperlipidemia and the development and progression of coronary artery disease and atherosclerosis. PUBLIC HEALTH RELEVANCE: Elevated levels of lipids and low density lipoprotein (LDL)-cholesterol, also known as the bad cholesterol, in plasma are major contributors to the development of coronary artery disease and atherosclerosis. Apolipoprotein B is the major protein component of all atherogenic lipoproteins and its levels in plasma predicate the risk for atherosclerosis. The goal of this application is to understand the structure function relationship of human apoB and to apply this information to the development of strategies aimed at attenuating the overproduction of hepatic apoB- containing lipoproteins and decreasing the risk of atherosclerosis.